This proposal aims to elucidate how the bacterial enzyme nitrogenase catalyzes the chemically difficult transformation of atmospheric dinitrogen into a bioavailable form, ammonia, and why/how it utilizes ATP hydrolysis to drive this reaction. Being the only enzyme responsible for reductive nitrogen fixation, nitrogenase sustains the agricultural/nutritional needs of ~40% of the human population. Aside from its global importance, nitrogenase is a unique model system with broad relevance to biological redox catalysis as well as ATP/GTP-dependent energy transduction processes, which are both central to proper cellular functioning and thus directly relevant to human health. Despite four decades of extensive biochemical, biophysical and structural characterization, the two most important questions about nitrogenase mechanism are not answered: a) Why and how ATP hydrolysis is ultimately utilized for the reduction of N2 or alternative substrates? b) What is the intimate mechanism of dinitrogen on the nitrogenase active site cluster, FeMoco? To make any further progress toward answering these questions, new experimental approaches and testable hypotheses are needed. Toward this end, a new strategy was developed to photochemically activate nitrogenase catalysis in the absence of ATP hydrolysis, which opens up new avenues to populate discrete catalytic intermediates on FeMoco for structural characterization. At the same time, the capability was acquired to rapidly generate site-directed mutants of nitrogenase proteins. Motivated by these advances, recent crystallographic findings, extensive experience on nitrogenase and collaborations with world-class spectroscopy laboratories, the PI and his group are uniquely positioned to address outstanding mechanistic issues in biological nitrogen fixation. The objectives of this project are to: 1) Determine the mechanistic role of multiple ATP-dependent docking interactions between the two nitrogenase components, the MoFe-protein (catalytic component) and the Fe-protein (ATPase/electron donor). The complex between the Fe-protein and MoFe-protein was structurally characterized in five distinct nucleotide states, whereby the Fe-protein populates several docking zones on the MoFe-protein surface. These docking zones are hypothesized to enable rapid successive one-electron transfer (ET) reactions to FeMoco to promote the 8- electron catalytic turnover, and will be subjected to systematic structure-function studies. 2) Identify the structural/electronic features of the MoFe-protein that are critical for controlling electron flow between its two Fe-S clusters, the P-cluster and FeMoco. Several lines of research have indicated the necessity of a conformational gate to enable electron flow from P-cluster to FeMoco for catalysis, which is hypothesized to be a protonation/deprotonation event. The nature of this gate will be probed through enzyme activity assays, photo-initiated ET and various spectroscopic techniques, using MoFe-protein variants with perturbed electron and proton transfer pathways. 3) Characterize the FeMoco structure in an activated/substrate-bound state. FeMoco can only bind substrates/inhibitors upon reduction beyond its as-isolated state under constant ATP turnover conditions. The newly developed photocatalytic scheme will be exploited to populate FeMoco in a one-electron reduced state primed for substrate binding, and the structures of ensuing intermediates will be characterized by crystallography and an array of spectroscopic techniques (EPR, NRVS, IR, Mssbauer). By meeting these project goals, the PI and his group will not only uncover the mechanistic details of this enzyme, but also provide general insights into biological multi-electron/proton redox catalytic processes and the transduction of ATP energy into chemical or mechanical work.